Modified Abstract. This research group recently published the first description of diagonal capillary electrophoresis. Diagonal capillary electrophoresis is a form of two-dimensional capillary electrophoresis that employs identical separation modes in each dimension. The distal end of the first capillary incorporates an enzyme-based microreactor. Analytes that are not modified by the reactor will have identical migration times in the two capillaries and will generate spots that fall on the diagonal in a reconstructed two-dimensional electropherogram. Analytes that undergo enzymatic modification in the reactor will have a different migration time in the second capillary and will generate spots that fall off the diagonal in the electropherogram. Dephosphorylation and desialylation result in the loss of a negative charge, which is trivial to detect using capillary electrophoresis. In this proposal, we will develop diagonal capillary electrophoresis as a robust tool for the determination of the phosphorylation status of complex samples. The technology offers significant advantages compared with alternatives. Most importantly, the technology provides accurate estimates of the extent of modification without artifact due to differences in ionization efficiency of the native and modified peptides. Also, the technology is fully automated: once the sample is loaded into the instrument, no fraction collection, pipetting, or other sample manipulations are performed. Finally, the technology employs capillary electrophoresis, which appears to be better suited than reversed-phase liquid chromatography for the analysis of peptides containing charged modifications.

Public Health Relevance

. The phosphorylation status of a protein modulates the activity of that protein, which is important in a wide range of diseases. This proposal provides the first fully automated method to identify phosphorylated peptides without interference.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM096767-04
Application #
8710257
Study Section
Enabling Bioanalytical and Biophysical Technologies Study Section (EBT)
Program Officer
Edmonds, Charles G
Project Start
2011-08-01
Project End
2015-07-31
Budget Start
2014-08-01
Budget End
2015-07-31
Support Year
4
Fiscal Year
2014
Total Cost
$285,000
Indirect Cost
$95,000
Name
University of Notre Dame
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
824910376
City
Notre Dame
State
IN
Country
United States
Zip Code
46556
Hayes, Michael H; Peuchen, Elizabeth H; Dovichi, Norman J et al. (2018) Dual roles for ATP in the regulation of phase separated protein aggregates in Xenopus oocyte nucleoli. Elife 7:
Sindelka, Radek; Abaffy, Pavel; Qu, Yanyan et al. (2018) Asymmetric distribution of biomolecules of maternal origin in the Xenopus laevis egg and their impact on the developmental plan. Sci Rep 8:8315
Qu, Yanyan; Sun, Liangliang; Zhu, Guijie et al. (2018) Sensitive and fast characterization of site-specific protein glycosylation with capillary electrophoresis coupled to mass spectrometry. Talanta 179:22-27
Zhang, Zhenbin; Dovichi, Norman J (2018) Optimization of mass spectrometric parameters improve the identification performance of capillary zone electrophoresis for single-shot bottom-up proteomics analysis. Anal Chim Acta 1001:93-99
Dai, Chen; Arceo, Jennifer; Arnold, James et al. (2018) Metabolomics of oncogene-specific metabolic reprogramming during breast cancer. Cancer Metab 6:5
Lee-Liu, Dasfne; Sun, Liangliang; Dovichi, Norman J et al. (2018) Quantitative Proteomics After Spinal Cord Injury (SCI) in a Regenerative and a Nonregenerative Stage in the Frog Xenopus laevis. Mol Cell Proteomics 17:592-606
Qu, Yanyan; Sun, Liangliang; Zhang, Zhenbin et al. (2018) Site-Specific Glycan Heterogeneity Characterization by Hydrophilic Interaction Liquid Chromatography Solid-Phase Extraction, Reversed-Phase Liquid Chromatography Fractionation, and Capillary Zone Electrophoresis-Electrospray Ionization-Tandem Mass Spectro Anal Chem 90:1223-1233
Peuchen, Elizabeth H; Cox, Olivia F; Sun, Liangliang et al. (2017) Phosphorylation Dynamics Dominate the Regulated Proteome during Early Xenopus Development. Sci Rep 7:15647
Flaherty, Ryan J; Sarver, Scott A; Sun, Liangliang et al. (2017) A High Voltage Power Supply That Mitigates Current Reversals in Capillary Zone Electrophoresis-Electrospray Mass Spectrometry. J Am Soc Mass Spectrom 28:247-252
Peuchen, Elizabeth H; Zhu, Guije; Sun, Liangliang et al. (2017) Evaluation of a commercial electro-kinetically pumped sheath-flow nanospray interface coupled to an automated capillary zone electrophoresis system. Anal Bioanal Chem 409:1789-1795

Showing the most recent 10 out of 64 publications